The present invention relates to a process for preparation of (R)-1,2-propanediol from racemic 1,2-propanediol, using a microorganism which has ability to assimilate (S)-1,2-propanediol as a single carbon source.
(R)-1,2-propanediol prepared by the present invention is very important and useful as an intermediate in preparing optically active compounds, such as pharmaceuticals, agrochemicals and physiologically active compounds.
In regard to a chemical method for preparation of an optically active 1,2-propanediol, a method by reduction of hydroxy acetone using BINAP catalyst by Kitamura et al. (Tetrahedron Lett., 32,4163-4166 (1991)) and a method by asymmetric hydrolysis using Co-Salen catalyst by Jacobsen et al. (Science, 277, 936-938 (1997) are known. But in order to prepare 1,2-propanediol with optically high purity by these methods, expensive chemical catalyst is necessary and therefore, these methods are hardly said to be an industrially economical method.
In regard to a biological method, a method for preparation of (R)-1,2-propanediol by asymmetric reduction of 1-hydroxy-2-propanone and 1-hydroxy-2-butanone using glycerol dehydrogenase is known (Journal of Organic Chemistry, 51, 25-36(1986)).
Further, a method of preparation for optically active 1,2-propanediol by stereoselectively oxidative degradation using resting cells of microorganisms belonging to a genus Pseudomonas strain by Nikaido et al. (Japanese patent publication A 6-30790). According to this method, as mentioned in the example thereof, after separately cultivating a large amount of microorganisms having ability to stereoselectively degrade 1,2-propanediol and changing thus obtained cells into resting cells, the cells must be subjected to the optical resolution.
The present inventors also reported a method of preparing optically active 1,2-diols and halogenohydrines using oxidative dehalogenation enzyme, namely halohydrin dehydro-dehalogenase derived from Alcaligenes sp. DS-S-7G (Tetrahedron: Asymmetry, 5, 239-246 (1994), Japanese patent publication A 7-147993).
However, in the above mentioned biological methods, co-enzyme such as NAD or NADP is necessary and the regeneration (recycling) of it becomes its reaction rate-limiting factor. These methods are not said to be an economical process for preparation of optically active 1,2-propanediol and therefore, the development of a more effective and practical method has been desired.
The object of the present invention is to provide a more economical and technically simpler method of preparing (R)-1,2-propanediol from racemic 1,2-propanediol comparing with the known methods mentioned above.
The present inventors have searched microorganisms having ability to assimilate (S)-1,2-propanediol preferentially and further, to grow by assimilating (S)-1,2-propanediol as a single carbon source. As a result the present inventors have succeeded in isolating the objective microorganisms and have completed the present invention.
Namely, the present invention relates to a process for preparation of (R)-1,2-propanediol which is characterized in cultivating a microorganism belonging to genus Pseudomonas or genus Alcaligenes which has ability to assimilate (S)-1,2-propanediol as a single carbon source, in a culture medium containing racemic 1,2-propanediol as a single carbon source or a complete synthetic medium containing racemic 1,2-propanediol as a single carbon source, assimilating (S)-1,2-propanediol and then isolating the remaining (R)-1,2-propanediol from the culture broth.
The microorganisms used in the present invention (abbreviated as the present microorganisms), for example Pseudomonas sp. DS-SI-5, Pseudomonas nitroreducens DS-S-RP8 and Alcaligenes sp. DS-S-7G have also ability to assimilate (R)-3-halogeno-1,2-propanediol and in case of the assimilation thereof, hydrogen halide of the same amount as (R)-3-halogeno-1,2-propanediol which is assimilated is released (Japanese patent publication B 4-73999, Japanese patent publication A 2001-149090). Thus, the present microorganisms are strains showing stereoselective dehalogenation ability against halogenohydrin compounds.
Furthermore, although Pseudomonas sp. DS-SI-5 has not ability to assimilate 4-chloro-1,3-butanediol, this strain shows stereoselective dehalogenation activity against (S)-4-chloro-1,3-butandiol to produce (S)-hydroxy-xcex3-butyrolactone (Japanese patent publication A 2001-120296).
Furthermore, Pseudomonas sp. DS-SI-5 does not have ability to assimilate 1,3-propanediol, 3-amino-1,2-propanediol, 1,2-butanediol, 1,2-pentanediol and 1,2-hexanediol.
Moreover, the present inventors have found that the present microorganisms have ability to stereoselectively assimilate (S)-1,2-propanediol, can grow in the complete synthetic medium containing racemic 1,2-propanediol as a single carbon source and makes non-assimilated (R)-1,2-propanediol remain in the culture broth.
The present invention relates to a process for preparation of (R)-1,2-propanediol from racemic 1,2-propanediol, by preferentially assimilating (S)-1,2-propanediol, using a microorganism belonging to genus Pseudomonas or genus Alcaligenes which has ability to assimilate (S)-1,2-propanediol as a single carbon source, and by isolating remaining (R)-1,2-propanediol from the culture broth.
Illustratively, first the present microorganisms are cultivated in the complete synthetic medium containing racemic 1,2-propanediol or 3-chloro-1,2-propanediol as a single carbon source, inorganic nitrogen compounds such as various ammonium salts or nitrates as nitrogen source and a small amount of metal salt or phosphate, or in a usual nutrient medium containing organic carbon subsatances and nitrogen substances such as a bouillon medium or a peptone medium and inorganic nutrient sources to prepare seed microorganisms.
Then, a small amount of thus obtained culture broth or microorganism is inoculated with a medium containing racemic 1,2-propanediol as a single source (abbreviated as the medium used in the present invention) and the medium is cultivated to isolate remaining (R)-1,2-propanediol from the culture broth.
Namely, by preferentially assimilating (S)-1,2-propanediol from racemic 1,2-propanediol using the present microorganisms, non-assimilated (R)-1,2-propanediol is made remain in the culture broth and is isolated.
The cultivation is preferably carried out at optimum pH and at optimum temperature, for example at pH 4-10, preferably 5-9, at the temperature of 15-50xc2x0 C., preferably 20-37xc2x0 C. In case that pH value gradually decreases with progress of the assimilation, it is necessary to adjust pH in the solution to optimum pH by adding an alkaline substance. The solution is preferably controlled in optimum pH range by using a substance which can be usually used as an acid-neutralizing agent, such as an aqueous alkali carbonate solution such as a calcium carbonate solution, a sodium carbonate solution, a potassium carbonate solution, or an ammonium carbonate solution, an aqueous alkaline hydroxide solution such as a sodium hydroxide solution, a potassium hydroxide solution or a calcium hydroxide solution, or an aqueous ammonia solution.
The concentration of the substrate, namely racemic 1,2-propanediol as carbon source in the medium is preferably 1-15% (v/v), and the substrate may be added at once in the initial stage or in several times.
The cultivation is usually aerobically carried out under stirring or agitation, or under aerobical agitating-cultivation, and the cultivation time depends on the substrate-concentration and other cultural condition, but is preferably completed in 24-120 hours.
When the remaining amount of the substrate, namely racemic 1,2-propanediol becomes 50% comparing with the initial concentration of the substrate by gas chromatography etc., the cultivation is preferably quenched, or the end point may be preferably determined with the measurement of optical purity on the optically active compound in the medium. Namely, exactly when (S)-1,2-propanediol in the substrate, namely racemic 1,2-propanediol is completely assimilated, the cultivation is preferably quenched.
Thus obtained (R)-1,2-propanediol remaining in the culture broth is recovered and purified by the conventional method. For example, after removal of cells from the medium by centrifugation, the supernatant is condensed with an evaporator, extracted with a solvent such as ethyl acetate etc. The extract is dried over anhydrous magnesium sulfate, and then the solvent is evaporated in vacuo to obtain (R)-1,2-propanediol in syrup. The syrup may be further purified by distillation.
In case of the practice of the present invention, the present microorganisms, without previous cultivation thereof, may be directly inoculated with the medium used in the present invention, and the medium is cultivated in the same manner as mentioned above to recover or isolate (R)-1,2-propanediol from the culture broth.
According to the present optical resolution method, the medium used is hardly contaminated as it is the complete synthetic medium containing racemic 1,2-propanediol as a single carbon source and that as (S)-1,2-propanediol is assimilated by the present microorganism, it is extremely easy to isolate (recover) remaining (R)-1,2-propanediol from the culture broth.
The medium used for previous seed culture is not limited as long as the present microorganisms can grow in said medium.
For example, there are illustrated carbohydrates such as glucose or fluctose, alcohols such as racemic 3-chloro-1,2-propanediol, (R)-3-chloro-1,2-propanediol, racemic 3-bromo-1,2-propanediol, (R)-3-bromo-1,2-propanediol or racemic 1,2-propanediol, organic acids such as acetic acid, citric acid, malic acid, maleic acid, fumaric acid, or gluconic acid, or a salt thereof, or a mixture thereof, as carbon source.
For example, there are illustrated inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, or ammonium phosphate, organic nitrogen compounds such as urea, peptone, casein, yeast extract, meat extract, corn steep liquor, or a mixture thereof, as nitrogen source.
Further, inorganic salts such as a phosphate, a magnesium salt, a potassium salt, a manganese salt, an iron salt, a zinc salt, a cooper salt, or if suitable, vitamins may be used.
As enzyme-inducing additives to prepare the microorganisms having high enzyme activity, to the above mentioned medium, a nutrient medium such as a peptone medium, or a bouillon medium may be added a racemic 3-halogeno-1,2-propanediol such as racemic 3-chloro-1,2-propanediol or racemic 3-bromo-1,2-propanediol, or racemic 1,2-propanediol.
The cultivation for preparing the seed culture can be carried out as in the usual manner, for example at pH 4-10, preferably 5-9, at the temperature of 15-50xc2x0 C., preferably 20-37xc2x0 C., and aerobically for 20-96 hours.
The present microorganisms are, as mentioned above, ones belonging to genus Pseudomonas and genus Alcaligenes which have ability to assimilate (S)-1,2-propanediol and can grow by assimilating (S)-1,2-propanediol as carbon source, and preferable ones are Pseudomonas sp. DS-SI-5, Pseudomonas nitroreducens DS-S-RP-8 and Alcaligenes sp. DS-S-7G.
These strains were identified as strains belonging to species or strains of genus Pseudomonas and genus Alcaligenes from their physiological and bacteriological properties and have been deposited with the International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology, Japan under Budapest Treaty under following deposit numbers.
Deposit number of Pseudomonas sp. DS-SI-5: FERM BP-7080, which has been deposited at the National Institute of Bio-Science and Human-Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan, on Oct. 7, 1999, under the Budapest Treaty; Deposit number of Pseudomonas nitroreducens DS-S-RP8: FERM BP-7793, which has been deposited at the International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology of AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan, on Jun. 29, 2001, under the Budapest Treaty; and Deposit number of Alcaligenes sp. DS-S-7G: FERM BP-3098, which has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan, on Nov. 15, 1989, under the Budapest Treaty.
Pseudomonas nitroreducens DS-S-RP8 (FERM BP-7793) is new strains which is not described in the publication and its physiological and bacteriological properties are shown below.
The present invention is illustratively explained by following examples, but should not be limited by these examples. Percentage (%) in examples means % (w/v), if not defined otherwise.